CULTURE MEDIUM FOR EPITHELIAL STEM CELLS AND ORGANOIDS COMPRISING THE STEM CELLS

• US 20140256037A1
• Filed: 11/13/2013
• Published: 09/11/2014
• Est. Priority Date: 02/03/2009
• Status: Active Grant

• Current Assignee: Koninklijke Nederlandse Akademie Van Wetenschappen
• Original Assignee: Koninklijke Nederlandse Akademie Van Wetenschappen
• Inventors: Sato, Toshiro, Clevers, Johannes C., Huch Ortega, Meritxell
• Granted Patent: US 10,947,510 B2
• Time in Patent Office: Days
• US Class Current: 435/371
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0679 Cells of the gastro-intestinal tract

Abstract

The invention relates to a method for culturing epithelial stem cells, isolated tissue fragments comprising said epithelial stem cells, or adenoma cells, and culturing the cells or fragments in the presence of a Bone Morphogenetic Protein (BMP) inhibitor, a mitogenic growth factor, and a Wnt agonist when culturing epithelial stem cells and isolated tissue fragments. The invention further relates to a cell culture medium comprising a BMP inhibitor, a mitogenic growth factor, and a Wnt agonist, to the use of said culture medium, and to crypt-villus organoids, gastric organoids and pancreatic organoids that are formed in said culture medium.

Claims

1. (canceled)

2. An in vitro method for obtaining a pancreatic organoid the method comprising:

incubating an epithelial stem cell or an isolated tissue fragment comprising an epithelial stem cell with an extracellular matrix; and

culturing the stem cell or isolated tissue fragment for a length of time sufficient to be able to observe formation of a pancreatic organoid, in an animal or human cell culture medium comprising;

a Bone Morphogenetic Protein (BMP) inhibitor,

between 5 and 500 ngram/ml of a mitogenic growth factor, and

a Wnt agonist, and obtaining from the culture medium a pancreatic organoid.

3. The method according to claim 2, wherein the BMP inhibitor is Noggin, the mitogenic growth factor is Epidermal Growth Factor (EGF), and the Wnt agonist comprises any one of R-spondin 1 through R-spondin 4.

4. The method according to claim 2, wherein the BMP inhibitor is selected from the group consisting of Noggin, DAN, Cerberus and Gremlin.

5. The method according to claim 2, wherein said Wnt agonist is selected from the group consisting of one or more of Wnt, R-spondin 1 through R-spondin 4, Norrin, and a GSK-inhibitor.

6. The method according to claim 2, wherein the Wnt agonist is one of R-spondin 1 through R-spondin 4 and Wnt-3a.

7. The method of claim 2, wherein the cell culture medium further comprises a Rock (Rho-kinase) inhibitor selected from the group consisting of Y-27632, Fasudil, and H-1152.

8. The method according to claim 2, wherein the cell culture medium further comprises a notch agonist.

21. The method according to claim 2, wherein the mitogenic growth factor is EGF, FGF and/or KGF.

22. The method according to claim 4, wherein the Wnt agonist is selected from the group consisting of one or more of Wnt, R-spondin 1 through R-spondin 4, Norrin, and a GSK-inhibitor.

23. The method according to claim 3, wherein the Wnt agonist comprises any one of R-spondin 1 through R-spondin 4 and Wnt-3a.

24. The method according to claim 4, wherein the Wnt agonist comprises any one of R-spondin 1 through R-spondin 4 and Wnt-3a.

25. The method according to claim 5, wherein the Wnt agonist comprises any one of R-spondin 1 through R-spondin 4 and Wnt-3a.

26. The method according to claim 4, wherein the mitogenic growth factor is EGF, FGF and/or KGF.

27. The method according to claim 5, wherein the mitogenic growth factor is EGF, FGF and/or KGF.

28. The method according to claim 3, wherein the cell culture medium further comprises a Rock (Rho-kinase) inhibitor selected from the group consisting of Y-27632, Fasudil, and H-1152.

29. The method according to claim 4, wherein the cell culture medium further comprises a Rock (Rho-kinase) inhibitor selected from the group consisting of Y-27632, Fasudil, and H-1152.

30. The method according to claim 5, wherein the cell culture medium further comprises a Rock (Rho-kinase) inhibitor selected from the group consisting of Y-27632, Fasudil, and H-1152.

31. The method according to claim 6, wherein the cell culture medium further comprises a Rock (Rho-kinase) inhibitor selected from the group consisting of Y-27632, Fasudil, and H-1152.

32. The method according to claim 3, wherein the cell culture medium further comprises a notch agonist.

33. The method according to claim 4, wherein the cell culture medium further comprises a notch agonist.

34. The method according to claim 5, wherein the cell culture medium further comprises a notch agonist.

35. The method according to claim 6, wherein the cell culture medium further comprises a notch agonist.

36. The method according to claim 7, wherein the cell culture medium further comprises a notch agonist.

37. The method according to claim 3, wherein the cell culture medium further comprises brain-derived neurotrophic factor.

38. The method according to claim 4, wherein the cell culture medium further comprises brain-derived neurotrophic factor.

39. The method according to claim 5, wherein the cell culture medium further comprises brain-derived neurotrophic factor.

40. The method according to claim 6, wherein the cell culture medium further comprises brain-derived neurotrophic factor.

41. The method according to claim 7, wherein the cell culture medium further comprises brain-derived neurotrophic factor.

42. The method according to claim 8, wherein the cell culture medium further comprises brain-derived neurotrophic factor.

44. The method according to claim 2, wherein the epithelial stem cell is an adenoma cell.

9. An in vitro method for obtaining and/or culturing a pancreatic organoid, the method comprising:

culturing epithelial stem cells or isolated tissue fragments comprising epithelial stem cells in contact with an extracellular matrix for a length of time sufficient to be able to observe formation of a pancreatic organoid, in a medium comprising Noggin, EGF and Keratinocyte Growth Factor or Fibroblast Growth Factor, any one of R-spondin 1 through R-spondin 4 and/or Wnt-3, B27, N2, and N-Acetylcystein; and

obtaining from the culture medium a pancreatic organoid.

19. The method according to claim 9, wherein the cell culture medium further comprises [Leu15]-Gastrin I, Exendin and/or Nicotinamide.

45. The method according to claim 9, wherein the epithelial stem cells are adenoma cells.

10-18. -18. (canceled)

20. An in vitro method for obtaining a pancreatic organoid, the method comprising:

culturing epithelial stem cells or isolated tissue fragments comprising epithelial stem cells on an extracellular matrix for a length of time sufficient to be able to observe formation of a pancreatic organoid, in an animal or human cell culture medium comprising;

a Bone Morphogenetic Protein (BMP) inhibitor;

a Wnt agonist; and

between 5 and 500 nanogram/ml of Epidermal Growth Factor (EGF); and

obtaining from the culture medium a pancreatic organoid.

46. The method according to claim 20, wherein the epithelial stem cells are adenoma cells.

43. An in vitro method for obtaining a pancreatic organoid, the method comprising:

incubating an epithelial stem cell or an isolated tissue fragment comprising an epithelial stem cell with an extracellular matrix, wherein the epithelial stem cell is an adenoma cell, and

culturing the stem cell or isolated tissue fragment for a length of time sufficient to be able to observe formation of a pancreatic organoid, in an animal or human cell culture medium comprising;

a Bone Morphogenetic Protein (BMP) inhibitor, and

between 5 and 500 ngram/ml of a mitogenic growth factor, and

obtaining from the culture medium a pancreatic organoid.